The intent of this project is to develop new and improve existing instrumentation and to develop new experimental and data analysis methods for the characterization of biological macromolecules and the study of their interactions. In the area of the analysis of the data from analytical ultracentrifugation, further refinements have been made in the major breakthrough achieved by the use of light intensity data rather than light absorbency data from the analytical ultracentrifuge. The intensity data is advantageous because of its superior statistical properties and its potential to provide better parameter estimates in ultracentrifugal analyses. The major contribution in this area has been the development of a particularly robust and statistically valid method for estimating weights to be used when fitting light intensity data by non-linear least-squares curve-fitting. An invited paper describing this method of estimated weights is in preparation for publication in the prestigious series Methods in Enzymology. Additional experience with this method has been gained, as it has been extensively applied to a variety of interacting systems studied in a variety of collaborative projects. In additiona, a new method of multi-wavelength analysis has been developed which facilitates analysis of protein-protein and protein-nucleic acid interactions. In this method, one or both reactants are uniquely labeled with a chromophore; this permits a more definitive observation of the behavior, both alone and associated, of that component in the analysis by mathematical modeling. This new method was utilized in Project 1 Z01 OD10039-05, "Physical Biochemistry of Macromolecules."